Hi, my name is Dr. Julia Nepper,
and today I’m going to be telling you
about how to model a soil microbiome.
So, I want you to think of the adage
“missing the forest for the trees”.
If you’re not familiar with this saying, w
hat it means is that you’re focusing so intently
on one particular aspect of something
that you’re missing the larger context.
Now, I don’t study trees.
I study soil bacteria.
But the underlying principle of understanding the larger context
is part of what I study.
There’s another thing that you may not think of
when you think about trees,
and that’s the soil and the roots.
And there’s another saying that’s common
— “dumber than dirt” —
and I’m going to show you today that soil is anything but stupid.
Far from being an inert mass of minerals,
soil is actually full of life.
There are larger creatures that you may be more familiar with,
like moles and earthworms,
but in addition there are billions and billions of microorganisms.
A single teaspoon of rich soil can have as many microorganisms,
as many bacteria,
as there are people in the United States,
so hundreds of millions of bacteria per teaspoon.
So, what we’re interested in doing with these bacteria
is forming what’s called a model system.
Model systems are very useful tools
for simplifying a problem and taking it to the lab
and doing experiments with it
that we can then extrapolate to a different, more complex system.
One that you may be familiar with is the common lab rat,
Rattus norvegicus domestica,
which we use as a model for understanding diseases in humans
as well as understanding rats.
And one of the reasons why we’re interested in this
is because we can often find emergent properties
when we look at systems in their entirety.
So, what this means, basically,
is that the individuals together are greater than the sum of their parts.
So, you can think of, for example, a heart.
Heart cells individually are not able to pump blood through our body,
but if you get billions of them together,
then you can form the organ the heart,
which has the emergent property of being able to pump blood.
Now, I told you that I was going to talk about soil bacteria,
but even that is a bit too large of a problem
to tackle with just one model.
So, what I’m actually going to be talking about today
is a zone specifically called the rhizosphere,
from the greek “rhizome”, meaning root.
You can think of this as the plant root microbiome.
It’s basically the area of soil directly on about about a millimeter away from the roots.
And this is the area of soil that’s directly impacted by plants.
One of the reasons why we’re very interested in the rhizosphere
is because of agricultural reasons.
If we can directly affect the rhizosphere,
then we can have effects on the plant
that can cause them to be more drought resistant
or to produce more fruit, etc.
Another reason why we’re interested in studying the rhizosphere
is because it can affect plants’ ability
to take up carbon dioxide
and form it into other carbon compounds,
which it uses to build itself.
So, if we can somehow alter the rhizosphere
so that the plants are better able to take up carbon dioxide,
then it’s possible that we could have some beneficial effect
on climate change.
So, I’m going to spoil a punch line a little bit
and I’m going to tell you what the model system is
before I tell you how we got there.
It’s called THOR — not the one you’re thinking of.
It stands for The Hitchhikers of the Rhizosphere,
and it’s a model system composed of three different bacteria:
pseudomonas koreensis,
flavobactermium johnsoniae,
and bacillus cereus,
which I’ll refer to as Pk, Fj, and Bc.
So, the first question, of course, is,
how did we create this system?
How did we get here?
Well, this started with some agricultural research,
in fact, about 30 years ago,
where researchers were trying to find biological agents
that could control diseases of alfalfa,
as opposed to chemical agents like pesticides.
The way they went about this is by isolating bacteria
directly from the roots of the alfalfa plants.
You can see the results of an example isolation from roots.
We get all of these individual colonies,
which are themselves propagations of an individual species
or strain of a species.
So, then we can select those colonies
and use them for further analysis.
So, the researchers isolated 700 different bacteria
from the roots of alfalfa.
They only found one that was able to prevent the disease
that they were looking at, and that was Bc.
But there was an issue when they actually took it back to the field
and tried to apply Bc to fields of alfalfa
to prevent this disease.
And that was that it was only somewhat effective —
sporadically effective.
And so they decided, well,
let’s look at what the larger context is of what this Bc
is existing in.
So, one thing that’s really interesting about Bc
is that it carries what’s called biological hitchhikers.
We’re not exactly sure why it does this,
but it could be because these bacteria
are using Bc to get somewhere
or using it for nutrients or something like that.
So basically, what a biological hitchhiker is…
is you will isolate the bacteria from roots,
as before,
and then you will take the individual colony that you’re interested in,
and you will attempt to purify it.
So, you’ll passage it on media a bunch of times,
until it looks like you just have that one particular bacterium.
And then you put it in the fridge for a couple days
and you come back to it,
and suddenly there’s a new species there.
There’s a new bacterium that’s growing
with what you thought was just Bc.
So then, we can take that species
and we can re-isolate the Bc
and isolate the new species,
and we can start to characterize both of them.
One hitchhiker that’s very common on Bc is Fj.
And it seems that the reason for this
is because Fj actually uses Bc
to be able to survive in the rhizosphere.
So, to imitate the rhizosphere environment in the lab,
use what’s called root exudate medium.
So, plants secrete a lot of molecules from their roots,
all the time.
And root exudate is composed of things like sugars, organic acids,
and other small molecules.
So, we take that and we make it into a medium
in which the bacteria can grow.
Bc grows just fine in this medium,
but Fj does not.
So, in the graph on the right, on the vertical y-axis
what we’re looking at is the number of Fj cells.
And on the horizontal x-axis,
we have different concentrations of cell wall fragments.
So, Bc will actually shed parts of its cell wall
into the medium,
and this is what actually feeds Fj.
So, you can see that as we get higher concentrations of cell wall fragments,
or, on the rightmost bar,
if we simply add Bc,
then Fj is suddenly able to grow in this medium,
indicating that it’s the cell wall fragments
that are feeding Fj in this medium.
There’s another hitchhiker that’s of particular interest to us in the lab,
and that’s Pk.
Interestingly enough,
Pk actually inhibits the growth of Fj
and other co-isolates that are closely related to it.
So, in this graph, again on the vertical y-axis,
we have number of bacterial cells.
And that’s not just of Fj —
it’s of whichever isolate is labeled on the bottom.
And then on the horizontal x-axis,
we have the different co-isolates,
and I’ve highlighted Fj for ease of viewing,
and then on the right Bc isn’t a hitchhiker,
but we’re just using it for comparison.
So, the blue bars are showing the amount of growth
that you get with these isolates
when they’re in isolation,
and then the orange bars are showing
the amount of growth that you get when you add Pk.
And you’ll see that there aren’t any orange bars
except in the case of Bc,
because Pk actually kills all of Fj
and its closely related co-isolates.
So, it was clear to us that these three species
were interacting in nature,
and interacting in interesting ways
that you wouldn’t expect if they were just
strangers in the night, passing each other.
So, the next question that we wanted to ask
after we established this system
is, how do these bacteria interact?
We need to characterize our system
so that we can use it better.
So, one of the first things that we noticed
is that Bc actually prevents Pk from killing Fj.
So, when we grow Fj in isolation,
in the blue line,
again it reaches a certain level,
as you can see on the vertical y-axis,
which is indicating the number of cells of Fj.
But if we add Pk, in the red line,
then you can see that over time,
as indicated on the horizontal x-axis,
Pk will completely kill all of the Fj,
until Fj is no longer detectable.
But if we add Bc to this mixture
— so, we have the full three-member community in one tube —
then Fj is able to grow just fine,
perhaps even slightly better than it would grow on its own.
And we found that the reason for this is an antibiotic set of molecules
called koreenceines
that are actually produced by Pk
in response to the presence of Fj.
But when we add Bc,
then Bc prevents the production of these koreenceines by Pk,
allowing Fj to live.
There’s another really interesting phenomenon
that occurs with the three-member community,
and that’s in biofilm formation.
So, biofilms are multicellular communities of unicellular organisms.
Something that you may be familiar with that is a biofilm
is rock slime in a pond or a lake.
So, a biofilm is the cells themselves,
and then a matrix of sugars and proteins
and other molecules that the organisms secrete
to keep themselves protected and together.
So, in the right image you can see a biofilm
composed of the three organisms,
with Pk labeled in blue, Fj in red, and Bc in green.
And you can see that they’re kind of stably associated with each other.
And the way that we measure this in the lab
is by growing these cells in what’s called a 96-well plate,
pictured on the left,
and then after a certain amount of time
we wash out any cells that are not adhering to the surface
and thus presumably not in the biofilm.
And then we can stain whatever material remains
with what’s called crystal violet.
And this is like the most exciting part of my week,
because most of the things that I do in the lab
are with clear, colorless liquids,
and so doing something that’s purple
is actually pretty great.
So then, once we’ve added the purple dye,
we can shoot light through each well,
and based on how much light passes through,
we have an indirect measure of how much biofilm there is.
So, I was interested in studying this phenomenon
when I came to the lab
because I studied biofilms when I was a graduate student.
And one thing that we wanted to know is
why this was happening.
So, Bc and Fj both cause Pk to form more biofilm.
So, on the vertical y-axis,
we have measures of biofilm
versus time on the horizontal x-axis.
And in the blue line, we have just Pk itself forming a biofilm.
It forms a certain amount,
and then over time that drops off as the biofilm begins to disperse.
Then, all of the other lines are Pk
plus either Bc or Fj or both.
And you can see that in all of these instances
Pk forms more biofilm than it would by itself.
So, we wanted to understand, why is this happening?
And at least in the case of Bc,
it seems to be at least partly because of this other antibiotic molecule,
called zwittermicin,
that Bc produces constitutively,
or all of the time.
So, we did a simple experiment
where we grew Pk with differing concentrations
of the zwittermicin,
which you can see on the horizontal x-axis.
And you can see that when we get
a certain concentration of the zwittermicin,
then the Pk biofilm formation almost doubles,
as you can see on the vertical y-axis,
relative to the level we reach without zwittermicin,
indicating that perhaps zwittermicin
is not just acting as a killing molecule,
as we usually expect for antibiotics,
but it may also be acting as a signaling molecule,
telling Pk, hey, I’m around,
let’s make a biofilm together.
So, that’s the hitchhikers of the rhizosphere,
two bacteria that hitchhike on this agriculturally relevant bacterium,
Bacillus cereus.
I’ve shown you that these bacteria
seem to interact in the real world.
So, Bc feeds Fj parts of its cell wall.
Fj causes Pk to produce koreenceines,
which in turn kill Fj.
Bc reduces koreenceine production by Pk,
allowing Fj to cohabitate with the other two.
And the zwittermicin produced by Bc
enhances the biofilm produced by Pk.
And altogether, these three organisms
form more biofilm than any of them would on their own.
Now, this is a lot to remember,
and I don’t expect you to remember all of these different phenomena
that I’ve shown you today.
But what I would like you to remember
is that these bacteria communicate with chemicals,
like many other bacteria.
So, we’ve seen koreenceines,
which have certain effects on these different bacteria,
and zwittermicin, which has a certain effect.
But there’s also many more chemicals
that we haven’t yet identified
that govern the interactions between all of these bacteria,
and bacteria in general.
So, what we’re interested in studying next is,
how does this model system actually interact with plants?
Because if you remember,
the rhizosphere is the plant root microbiome.
So, of course we don’t want to just understand
how the bacteria interact with each other;
we want to know how they actually interact with the plants.
So, in this image we can see
a plant root tip that is being colonized by Bc,
which is shown in green.
So, examples of questions that we might ask next are,
how does adding Pk or Fj affect Bc’s ability
to colonize a plant root?
Does it change the way in which Bc colonizes plant roots?
Does it change the way in which Bc affects the plant growth, etc?
And all of this is so that we can
identify basic principles of how microbiomes function,
not just for understanding plant microbiomes,
but also hopefully to understand human microbiomes,
cow gut microbiomes,
and all of the microbiomes that surround us constantly.
And specifically, we want to understand
how these organisms interact
because, again, nothing exists in isolation.
There is not just a tree;
there’s always a forest that we have to consider.
Thank you.